Hedgehog Signaling Controls Dorsoventral Patterning, Blastema Cell Proliferation and Cartilage Induction During Axolotl Tail
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Research article 3243 Hedgehog signaling controls dorsoventral patterning, blastema cell proliferation and cartilage induction during axolotl tail regeneration Esther Schnapp1, Martin Kragl1, Lee Rubin2 and Elly M. Tanaka1,* 1Max Planck Institute for Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany 2Curis Incorporated, 61 Moulton Street, Cambridge, MA 02138, USA *Author for correspondence (e-mail: [email protected]) Accepted 12 May 2005 Development 132, 3243-3253 Published by The Company of Biologists 2005 doi:10.1242/dev.01906 Summary Tail regeneration in urodeles requires the coordinated Proliferation of tail blastema cells was severely impaired, growth and patterning of the regenerating tissues types, resulting in an overall cessation of tail regeneration, and including the spinal cord, cartilage and muscle. The blastema cells no longer expressed the early cartilage dorsoventral (DV) orientation of the spinal cord at the marker Sox9. Spinal cord removal experiments revealed amputation plane determines the DV patterning of the that hedgehog signaling, while required for blastema regenerating spinal cord as well as the patterning of growth is not sufficient for tail regeneration in the absence surrounding tissues such as cartilage. We investigated this of the spinal cord. By contrast to the cyclopamine effect on phenomenon on a molecular level. Both the mature and tail regeneration, cyclopamine-treated regenerating limbs regenerating axolotl spinal cord express molecular markers achieve a normal length and contain cartilage. This study of DV progenitor cell domains found during embryonic represents the first molecular localization of DV patterning neural tube development, including Pax6, Pax7 and Msx1. information in mature tissue that controls regeneration. Furthermore, the expression of Sonic hedgehog (Shh) is Interestingly, although tail regeneration does not occur localized to the ventral floor plate domain in both mature through the formation of somites, the Shh-dependent and regenerating spinal cord. Patched1 receptor expression pathways that control embryonic somite patterning and indicated that hedgehog signaling occurs not only within proliferation may be utilized within the blastema, albeit Development the spinal cord but is also transmitted to the surrounding with a different topography to mediate growth and blastema. Cyclopamine treatment revealed that hedgehog patterning of tail tissues during regeneration. signaling is not only required for DV patterning of the regenerating spinal cord but also had profound effects on Key words: Axolotl, Regeneration, Sonic hedgehog, Cyclopamine, the regeneration of surrounding, mesodermal tissues. Blastema, Sox9, Pax7 Introduction by Holtzer strongly suggests that cartilage is induced by the Axolotl tail regeneration involves the regrowth and patterning ventral half of the spinal cord during urodele tail regeneration. of multiple tissue types, including the spinal cord, muscle, It also implies that the mature spinal cord maintains DV cartilage, dermis, fin and skin. After tail amputation and patterning information and that this patterning information is epithelial wound healing, the spinal cord grows out as a tube transmitted into the regenerate. Similarly, studies in limb of neuroepithelial progenitor cells, called the ependymal tube. regeneration indicate the presence of DV patterning The regenerating spinal cord is surrounded by blastema cells, information within the mature limb that is required for proper the progenitor cells that give rise to the mesodermal tissues in growth and patterning of the regenerate (Carlson, 1974; the tail, such as dermis, cartilage and muscle. How coordinated Carlson, 1975; Holder et al., 1980). growth and patterning of the different tissue types occurs on a While the molecular mechanisms underlying spinal cord molecular level during axolotl tail regeneration is still regeneration are poorly understood, the patterning of the unknown. Spinal cord transplantation experiments have developing neural tube into distinct DV progenitor domains has established that the spinal cord harbors crucial dorsoventral been molecularly characterized in recent years (reviewed by (DV) patterning information, not only for the regenerating Bronner-Fraser and Fraser, 1997; Ericson et al., 1997a; Tanabe spinal cord but also for the surrounding tissues such as muscle and Jessell, 1996). The neural tube is subdivided into distinct and cartilage (Holtzer, 1956). Holtzer rotated a piece of tail domains, as defined by a series of homeodomain and paired spinal cord 180° about its DV axis, implanted it back into the box-containing transcription factors, with the dorsalmost mature tail and amputated the tail through the operated region. domain defined by Msx1 and 2 expression, dorsolateral cells In this situation both the spinal cord and the surrounding tissue by Pax7, and lateral domains by Pax6, while Nkx6.1 and regenerated upside down, with cartilage forming dorsally with Nkx2.2 define increasingly ventral domains. The size and respect to the whole animal, but still next to the original ventral placement of these domains is controlled by several side of the spinal cord (Holtzer, 1956). This and further work morphogens. Sonic hedgehog (Shh), a cholesterol-modified 3244 Development 132 (14) Research article extracellular signaling factor expressed in the notochord and through the drug cyclopamine, we show that it is required not the floor plate, induces ventral neural tube cell types in a only for DV patterning of the spinal cord, but also for overall concentration-dependent manner (Briscoe et al., 1999; Ericson tail regeneration. Specifically, the proliferation of blastema et al., 1997a; Ericson et al., 1997b; Litingtung and Chiang, cells and Sox9 expression in the ventral blastema is dependent 2000; Roelink et al., 1995). The Shh gradient in the neural tube on hedgehog signaling. Therefore the induction of cartilage by is antagonized by dorsally secreted bone morphogenetic the spinal cord during tail regeneration is mediated at least in proteins (Bmps) from the epidermal ectoderm and the dorsal part through hedgehog. roof plate cells of the neural tube (Liem et al., 1995), which specify a subset of interneurons in the dorsal neural tube (Lee et al., 2000; Liem et al., 1997). Whereas Bmp4 and Bmp7 Materials and methods activate the expression of Msx1, Pax7 and Pax6 in the dorsal Axolotl care and lateral neural tube, Shh has a concentration-dependent Ambystoma mexicanum (axolotls) were bred in our facility, where they inhibitory effect on the expression of these markers (Goulding were kept at 18°C in Dresden tap water and fed daily with artemia. et al., 1993; Liem et al., 1995; Timmer et al., 2002). Low For all surgery, animals were anesthetized in 0.01% ethyl-p- concentrations of Shh block Msx1 and Pax7 expression but can aminobenzoate (Sigma). The experiments described here were elevate Pax6 expression in lateral neural tube cells. High performed on 3-cm-long larval axolotls. concentrations of Shh, however, inhibit Pax6 expression in In situ hybridization on axolotl tail cryosections and floor plate cells of the neural tube (Ericson et al., 1997b). Thus, sequences of probes used during embryogenesis, the notochord ventrally and the Axolotl tail tissue was fixed in 4% fresh paraformaldehyde (PFA) ectoderm dorsally impose DV patterning on the neural tube overnight at 4°C, washed in PBS, equilibrated in 30% sucrose and through extracellular signaling. embedded in tissue-tek (O.C.T. compound, Sakura). Cryosections 16 During development, the action of Shh and Bmps is not µm thick were mounted on Superfrost adhesive slides and dried at restricted to patterning the neural tube. These morphogens also room temperature (RT) for several hours. The sections were quickly play important roles in controlling cell proliferation, patterning washed in PBS and treated with hybridization denaturation mix (2% ϫ and cell-type specification of somite-derived cells such as the SDS, 100 mmol/l DTT in 1 PBS) for 20 minutes at RT. After three sclerotome, resulting in a coordinated patterning of the neural washes in PBS/0.1% Tween, the sections were digested with Proteinase K (2-10 µg/ml) for 5 minutes and post-fixed directly afterward with tube and its surrounding mesodermal structures. Shh mutant PFA for 10 minutes at RT. Slides were washed in PBS/Tween and mice lack vertebral columns and ribs, demonstrating that Shh incubated at RT for 15 minutes in triethanolamine with 0.25% acetic signaling from the notochord and ventral neural tube is crucial anhydride. After several washes in PBS/Tween, slides were for sclerotome development (Chiang et al., 1996). More prehybridized in hybridization buffer (50% formamide, 5ϫ SSC, 5ϫ specifically, Shh induces the expression of sclerotomal markers Denhardts, 750 µg/ml yeast RNA) for 1 hour at 68°C, and then such as Pax1 and Sox9 (Fan and Tessier-Lavigne, 1994; hybridized overnight at 68°C with 500 ng/ml DIG-labeled probe in Development Marcelle et al., 1999; Murtaugh et al., 1999; Zeng et al., 2002), hybridization solution. Slides were washed twice an hour at 68°C in which are essential for sclerotome development and cartilage post-hybridization solution (50% formamide, 2ϫ SSC, 0.1% Tween) ϫ formation (Bi et al., 1999; Peters et al., 1999). Similarly, Shh and then 3 10 minutes at RT in maleic acid buffer (100 mmol/l maleic regulates myogenic precursors by positively regulating Myf5 acid pH 7.5, 150 mmol/l NaCl, 0.1% Tween). Sections were